Targeted recombinant viral vectors

ABSTRACT

The present invention provides methods and a composition for targeted delivery of a nucleic acid to a cell comprising a biotinylated recombinant encapsidated virus, particularly adenovirus, wherein the recombinant adenovirus comprises the nucleic acid, and wherein the biotinylated recombinant virus is linked via streptavidin to a biotinylated targeting moiety.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is in the field of targeted gene delivery.Specifically, the invention relates to recombinant viral vectors fortargeted delivery to selected cells, wherein the recombinant virus is asmall, encapsidated virus, such as adenovirus or adeno-associated virus.

[0003] 2. Background Art

[0004] Recent attempts to target gene transfer to human cells havefocused on the use of retroviral and adenoviral vectors. However, themost promising current vectors have resulted in limited success, due inpart to the inability to target specific cell types such ashematopoietic stem cells, and the need to culture target cells in vitroto promote cell cycling which can result in the loss of stem cellfunction. The ability to target gene transfer to specific cell types insitu would greatly enhance current approaches to gene therapy.

[0005] Several approaches have been taken to target viral and non viralvectors that include using ligands, antibodies or peptides in the vectorconstruction thereby redirecting virus infection through antigens orreceptors expressed on specific cell types. Early experiments designedto redirect the host range of retroviruses by molecular modification ofecotropic envelopes to recognize cellular receptors through antigenbinding, ligand or peptide sequences were unsuccessful. It hassubsequently been shown that efficient uncoating of the virus requires aconformational change in a subunit of the envelope protein which is onlyinduced upon interaction of the retrovirus particle with its cognatereceptor; thus retroviruses directed to alternate receptors do not yieldhigh frequencies of infection.

[0006] Similarly, the use of bifunctional antibodies which recognizeboth viral epitopes and target cell antigens to redirect the tropism ofamphotropic retroviruses have not resulted in targeted gene transfer.Successful targeting of retroviruses has required construction ofchimeric amphotropic envelopes which contain a protease cleavage siteand a targeting moiety. Following recognition of the target cell by thetargeting moiety, proteases cleave the chimeric sequences to leave theintact amphotropic envelope. Because the virus is now in close proximityto the cell surface, the viral envelope can interact with its authenticreceptor and viral infection will proceed.

[0007] The present invention utilizes the biology of adenovirusinfections to confer several advantages to the use of recombinantadenoviruses for targeted gene transfer. Infection of a cell byadenovirus requires two distinct receptors, one for recognition and oneto mediate internalization of the virus. First, the knob portion of thecapsid fiber interacts with a recently identified receptor on the cellsurface, then internalization of the adenovirus is mediated byinteraction of the penton base protein with integrins (fibronectin andvitronectin) on the cell surface. The adenovirus capsid uncoats in theacidic endosome (i.e., does not require interaction with a receptor tomediate a conformational switch) and the DNA is delivered to the nuclearpore.

[0008] It has been demonstrated that targeting of adenoviruses can beaccomplished by redirection of host range by linkage to bispecificantibodies. Several groups have also reported that molecularmodifications to introduce chimeric viral proteins have met withsuccess. However, bispecific antibodies limit the usefulness of targetedvectors for in situ gene transfer due to the effects of serum onantigen-antibody interactions, and introduction of sequences encodingchimeric viral proteins into the viral genome can adversely affect virusfunction. Therefore, there is a need for more efficient, effective,specifically targeted adenoviral vectors that retain their infectivitycharacteristics for gene delivery for a variety of purposes.

[0009] The present invention fills this need by providing a vector, andparticularly an adenoviral vector, that can be linked to any selectedtargeting moiety for targeting to any selected cell type. The vector ishighly efficient, achieving high levels of expression of the transferredgene specifically in the targeted cells, The present invention providesa vector wherein biotin is covalently linked to recombinant adenovirusparticles such that through an avidin bridge we could redirect the virususing biotinylated growth factors and antibodies.

[0010] Targeted gene transfer to specific populations of hematopoieticprogenitor cells represents an advance towards therapeutic use of genetransfer. The c-kit receptor has been shown to be expressed on thesurface of primitive hematopoietic cells with long term reconstitutionactivity, and represents an attractive receptor for targeted genetransfer. Therefore, a molecular conjugate vector consisting of plasmidDNA encoding the luciferase reporter gene complexed with polylysinecoupled to avidin (for addition of the biotinylated ligand) anddefective adenovirus particles was used to target hematopoieticprogenitors (Blood 87:472-478, 1996). Upon the addition of biotinylatedsteel factor (SLF) we demonstrated that this vector specifically targetsc-kit⁺ hematopoietic cell lines and results in up to a ten fold increasein luciferase activity as compared to a control vector. Furthermore,transient gene expression is observed with maximum expression at 30hours, and greater than 90% of target cells are transfected. However,the potential for use of this vector for in vivo gene transfer islimited by the inclusion of polylysine in the molecular conjugate fortwo reasons: 1) polylysine yields a high background due to non-specificelectrostatic interactions with the cell surface, 2) the electrostaticcharges required to hold the molecular conjugate vector together may berapidly neutralized in the presence of human serum. Other laboratorieshave approached targeted gene transfer by modifying the tropism ofrecombinant adenoviral vectors through a neutralizing anti-fiberantibody chemically conjugated to cell-specific ligands (NatureBiotechnology 14:1574-1578, 1996). These vectors represent animprovement over the molecule conjugate vector in that the targetingmoiety is no longer attached to the vector through electrostaticcharges, resulting in a decrease in the non-specific background, but theaffinity of the antibody-antigen interaction required for targeting ofthese vectors is variable and is not stable in the presence of serum. Athird approach to targeted adenoviral vectors has been to molecularlyclone sequences encoding the targeting moiety into the fiber or pentonbase structural genes of the adenoviral vector. This approach isexpected to avoid the disadvantages of targeting throughantibody-antigen interactions, but necessitates extensive manipulationof the adenoviral genome to introduce each targeting moiety.

[0011] The present invention provides more efficient vectors for genetransfer. The vectors were generated by direct high affinity linkage ofthe targeting moiety to a recombinant virus particle. To achieve theseinventive vectors, the invention includes a protocol for the covalentaddition of biotin to the capsid of recombinant encapsidated small virusparticles. The present invention demonstrates that biotin can be linkedto the capsid of recombinant adenovirus while maintaining wild-typeinfectivity. Following incubation with streptavidin, any biotinylatedligand or antibody could be added to target this vector to any celltype.

SUMMARY OF THE INVENTION

[0012] The present invention provides a composition for targeteddelivery of a nucleic acid to a cell comprising a biotinylatedrecombinant encapsidated virus, wherein the recombinant virus comprisesthe nucleic acid to be delivered, and wherein the biotinylatedrecombinant virus is linked via streptavidin to a biotinylated targetingmoiety.

[0013] The present invention specifically provides a composition fortargeted delivery of a nucleic acid to a cell comprising a biotinylatedrecombinant adenovirus, wherein the recombinant adenovirus comprises thenucleic acid to be delivered, and wherein the biotinylated recombinantadenovirus is linked via streptavidin to a biotinylated targetingmoiety.

[0014] The present invention further provides a method for targeteddelivery of a nucleic acid to a selected cell in a subject comprisingadministering to the subject a composition comprising a biotinylatedrecombinant encapsidated virus comprising the nucleic acid to bedelivered, wherein the virus is linked via streptavidin to abiotinylated targeting moiety that specifically targets the selectedcell.

[0015] The present invention further provides a method for targeteddelivery of a nucleic acid to a selected cell in a subject comprisingadministering to the subject a composition comprising a biotinylatedrecombinant adenovirus comprising the nucleic acid to be delivered,wherein the virus is linked via streptavidin to a biotinylated targetingmoiety that specifically targets the selected cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the effects of concentration of biotinylated SLF onefficiency of targeted transfection by recombinant adenovirus.Hematopoietic cell lines were transfected wih the SLF-targetedrecombinant adenovirus encoding luciferase, and after 24 hours,harvested and luciferase gene expression measured. The graph depictsrelative light units (RLU) per mg protein as concentration of SLF isincreased from 0 to 1000 ng/ml, in c-kit⁺ cells. The data are presentedas the mean RLU of duplicate determinations±the SE and arerepresentative of at least three experiments.

[0017]FIG. 2 shows the effects of concentration of neutravidin ontargeted adenovirus transfection of MO7e cells. Cells were harvestedafter 48 hours. The graph shows the relative light units (RLU) per mgprotein measured from MO7e cells upon transfection with steelfactor-targeted recombinant adenovirus encoding luciferase, when variousconcentrations of neutravidin are used (from 0 to 40 μg/ml). The openbars represent control vector (non-targeted recombinant adenovirus:adenovirus vector linked to avidin without steel factor (complete vectorlacking targeting moiety)), and the closed bars represent completevector (with targeting moiety) of the present invention.

[0018]FIG. 3 shows the specificity of targeted adenovirus gene transferto MO7e cells through the c-kit receptor. Cells were transfected withthe recombinant adenovirus vector encoding luciferase having thetargeting moieties as indicated, harvested after 24 hours, andluciferase gene expression in the cells measured. The graph depictsrelative light units (RLU) per mg protein. The results indicate that SLFpresent but not linked to the adenovirus vector competed out the vectorfor targeting MO7e cells, and the addition of IL-3, an unrelatedtargeting moiety, did not compete for targeting MO7e cells. The firstset of bars is the negative control which is untreated MO7e cells. Theremaining bars are labeled as follows: No avidin: MO7e cells treatedwith biotinylated adenovirus in the absence of avidin and biotinylatedSCF; No avidin+SCF: MO7e cells treated with biotinylated adenovirus andbiotinylated SCF in the absence of avidin; Avidin: MO7e cells treatedwith biotinylated adenovirus linked to avidin in the absence ofbiotinylated SCF; Avidin+SCF: MO7e cells treated with biotinylatedadenovirus linked to biotinylated SCF through an avidin bridge;Avidin+cold SCF: MO7e cells pretreated with 500 ng/ml SCF weresubsequently infected with biotinylated adenovirus linked to avidin;Avidin+SCF+cold: MO7e cells pretreated with 500 ng/ml SCF weresubsequently infected with biotinylated adenovirus linked tobiotinylated SCF through an avidin bridge; Avidin+IL-3: MO7e cellspretreated with 500 ng/ml IL-3 were subsequently infected withbiotinylated adenovirus linked to avidin; and Avidin+IL-3+SCF: MO7ecells pretreated with 500 ng/ml IL-3 were subsequently infected withbiotinylated adenovirus linked to biotinylated SCF through an avidinbridge.

[0019]FIG. 4 shows the versatility of biotinylated recombinantadenovirus in targeting surface markers on MO7e cells. The open barsrepresent control vector, and the closed bars represent targeted vector.“Bio CD34”, “Bio CD44”, and “Bio CD117” represent recombinant adenovirusvector having as targeting moieties biotinylated antibodies to variouscell surface markers present on MO7e cells to which SLF can bind. Thegraph shows transfection (RLU/mg protein) of MO7e cells with recombinantadenovirus vectors having the indicated targeting moiety linked viabiotin-streptavidin linkage. The Y-axis is labeled as follows: 1e+4=10⁴;1e+5=10⁵; 1e+6=10⁶; 1e+7=10⁷; 1e+8=10⁸. The X-axis is labeled asfollows:

[0020] No Ligand: Open bar: MO7e cells infected with biotinylatedrecombinant adenovirus.

[0021] Closed bar: MO7e cells infected with biotinylated recombinantadenovirus treated with avidin.

[0022] +Bio SCF: Open bar: MO7e cells treated with biotinylatedrecombinant adenovirus and 25 ng of biotinylated SCF in the absence ofan avidin bridge. Closed bar: MO7e cells treated with biotinylatedrecombinant adenovirus linked to 25 ng of biotinylated SCF through anavidin bridge.

[0023] +Bio CD34: Open bar: MO7e cells treated with biotinylatedrecombinant adenovirus and biotinylated anti-CD34 antibody in theabsence of an avidin bridge. Closed bar: MO7e cells treated withbiotinylated recombinant adenovirus linked to biotinylated anti-CD34antibody through an avidin bridge.

[0024] +Bio CD44: Open bar: MO7e cells treated with biotinylatedrecombinant adenovirus and biotinylated anti-CD44 antibody in theabsence of an avidin bridge. Closed bar: MO7e cells treated withbiotinylated recombinant adenovirus linked to biotinylated anti-CD44antibody through an avidin bridge.

[0025] +Bio CD117: Open bar: MO7e cells treated with biotinylatedrecombinant adenovirus and biotinylated anti-CD117 (anti-c-kit) antibodyin the absence of an avidin bridge. Closed bar: MO7e cells treated withbiotinylated recombinant adenovirus linked to biotinylated anti-CD117(anti-c-kit) antibody through an avidin bridge.

[0026]FIG. 5 shows the kinetics of gene expression. The -•- indicatescontrol vector and -▪- indicates SLF-targeted recombinant adenovirusvector encoding luciferase. Expression of the luciferase gene ismeasured over 14 days (RLU/mg protein) following transfection.

[0027]FIG. 6 shows electron micrographs of (A) control and (B)biotinylated recombinant adenovirus. Recombinant adenovirus AdCMVuc wasincubated with 100 μg/mL photoactivatable biotin and irradiated at awavelength of 350 nm for 5 minutes; control virus was treatedidentically except for addition of photoactivatable biotin. Followingincubation With colloidal gold-streptavidin, negative stainimmunoelectron microscopy was performed (17) and photographs were taks amagnification of 135,000.

[0028]FIG. 7 shows (A) Infection of c-kit⁺ and c-kit⁺ cells bySCF-targeted recombinant adenovirus. Biotinylated recombinant adenoviruslinked to avidin was incubated with either 250 ng/mL of SCF (controlvector) or 250 ng/mL of bio-SCF (SCF-targeted vector) (18) prior toinfection of the c-Kit⁺ cell lines MO7e and MBO2 and the c-Kit⁺ celllines HL-60 and KG-1 (19). Twenty four hours after infection theluciferase activity was measured and reported as he average relativelight units (RLU)/mg protein of two determinations±SE. The results arerepresentative of two separate experiments. (B) Specificity ofSCF-targeted gene transfer to MO7e cells. SCF-targeted vectors wereconstructed by incubating biotinylated virus linked to avidin with 50 to1000 ng/mL of recombinant human bio-SCF. In competition experiments, theSCF-targeted vector was constructed by incubating biotinylated viruswith 250 ng/mL of bio-SCF, and MO7e cells were incubated with 500 ng/mLnon-biotinylated SCF or IL-3 prior to infection with 100 μL theSCF-targeted vector. (C) Kinetics of luciferase gene expressionfollowing infection of MO7e cells with SCF-targeted vectors.SCF-targeted vectors were constructed by adding 250 μg/mL tobiotinylated virus linked to avidin. At various times followinginfection, MO7e cells were harvested and assayed for luciferaseexpression. At all time points, luciferase activity of cells infectedwith the control vector was less than 2×10⁴ RLU/mg protein.

[0029]FIG. 8 shows frequency of SCF-targeted infection of MO7e cells.MO7e cells were either (A) untreated, (B) treated with 100 μL ofbiotinylated virus, or (C) treated with 100 μL of the SCF-targetedvector constructed with 250 ng/mL of bio-SCF. Twenty four hours later,cells were harvested and in situ PCR specific for amplification ofluciferase DNA was performed. Photographs were taken at 1000×magnification on a Leitz Laborlux D Wild MPS 46 microscope, and arerepresentative of 500 cells per slide scored on duplicate slides fromthree separate experiments.

[0030]FIG. 9 shows targeting of hematopoietic cell lines withbiotinylated recombinant adenovirus linked to antibodies. (A) Thec-kit⁺, CD34⁺, CD44⁺ cell line MO7e was infected with 100 μL ofbiotinylated recombinant adenovirus lined to avidin (control), 250 ng/mLbiotinylated SCF (SCF-targeted), 20 μg/mL biotinylated anti-CD117(antibody to c-kit, CD117-targeted), 20 μg/mL biotinylatedanti-CDer-targeted) or 2.5. μg/mL biotinylated anti-CD44 antibodies(CD44-targeted). The (B) c-kit⁺, CD34⁺, CD44⁺KG-1 cells and (C) c-kit⁺,CD34⁺, CD44⁺HL-60 cells were treated identically to MO7e cells.Luciferase activity at 24 hours after infection is reported as themean±SE of duplicate determinations and is representative of twoexperiments.

[0031]FIG. 10 shows the successful gene transfer to primary human Tcells using IL-2 and CD44 directed virus.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention is more particularly described in thefollowing examples which are intended as illustrative only sincenumerous modifications and variations therein will be apparent to thoseskilled in the art.

[0033] As used in the specification and in the claims, “a” can mean oneor more, depending upon the context in which it is used. “SLF” and “SCF”are used interchangeably to refer to steel factor.

[0034] The invention provides a vector for targeted delivery of anucleic acid to a cell comprising a biotinylated encapsidated virus,wherein the virus is linked via streptavidin to a biotinylated targetingmoiety. The encapsidated virus can be recombinant and can comprise thenucleic acid to be delivered to the cell. The vector can be in a cell,either in vivo or in vitro.

[0035] The present vectors were generated by direct high affinitylinkage of the targeting moiety to a recombinant adenovirus particle. Toachieve these inventive vectors, we developed a protocol for thecovalent addition of biotin to the capsid of recombinant adenovirusparticles. We have demonstrated that biotin can be linked to the capsidof recombinant adenovirus while maintaining wild-type infectivity.Following incubation with streptavidin, any biotinylated ligand orantibody could be added to target this vector to any cell type.Following incubation of this vector with biotinylated SLF, up to a onethousand fold increase in luciferase gene expression was observed.Expression of the transferred gene was observed over 96 hours.Versatility of the vector of this invention was demonstrated bysubstituting targeting moieties: antibodies to four different cellsurface markers for SLF, which resulted in targeted gene transfer tocell lines expressing those specific markers. Thus the present inventionprovides vectors and methods that can achieve targeted transientexpression of nucleic acids, such as cytotoxic genes, tumor-specificribonucleases and growth suppressor. These vectors can be utilized fortherapeutic applications, such as treatment of leukemic cells, treatmentof solid tumors in any cancer that expresses a tumor-specific cellsurface marker, such as breast cancer, lung cancer, cervical cancer,pancreatic cancer, liver cancer, colon cancer, prostate cancer andmelanoma. The invention can also be used to treat viral and bacterialinfections and autoimmune diseases.

[0036] Thus, the present invention provides a composition for targeteddelivery of a nucleic acid to a cell comprising a biotinylatedrecombinant encapsidated virus, wherein the recombinant virus comprisesthe nucleic acid, and wherein the biotinylated recombinant virus islinked via streptavidin to a biotinylated targeting moiety. The methodcan be used with small, encapsidated viruses. Examples of virus caninclude adenovirus and adeno-associated virus (AAV). Choice of viralvector can vary depending upon the purpose of the gene transfer. Forexample, for transient expression of the transferred gene, adenoviralvectors would be useful; for longer term expression, AAV vectors wouldbe useful.

[0037] Thus, in one example, the present invention provides acomposition for targeted delivery of a nucleic acid to a cell comprisinga biotinylated recombinant adenovirus, wherein the recombinantadenovirus comprises the nucleic acid, and wherein the biotinylatedrecombinant adenovirus is linked via streptavidin to a biotinylatedtargeting moiety. The targeting moiety can be, for example, a ligand, anantibody, e.g., an antibody directed against cell surface markers forsteel factor (such as anti-CD117 antibodies) or other antibodies (e.g.,anti-CD34, anti-CD44, anti-CD45, anti-CD5, anti-CD3, anti-CD4, anti-CD8,and anti-LFA-1). The molecule on the cell targeted by the targetingmoiety can be any selected targeted moiety, such as a cell surfacereceptor, such as a receptor that binds steel factor (c-kit). To targethematopoetic cells, for example, the receptor for steel factor (c-kit)can be targeted with an anti-c-kit antibody or it can be targeted withthe steel factor itself linked by biotin-avidin-biotin to the virus withsuccess at above of 99%. The antibodies, anti-CD45, anti-CD5, anti-CD3,anti-CD4, anti-CD8, and anti-LFA-1, are shown herein to be particularlyeffective at targeting primary T cells and NK cells. The vectors may beused for a variety of purposes, such as antisense delivery vectors, genetherapy vectors, and vaccines.

[0038] Steel factor can be soluble or membrane associated and isdescribed in Godin et al. (Effects of the steel gene product on mouseprimordial germ cells in culture, Nature 352, 807-809, 1991), Dolci etal. (Requirement for mast cell growth factor for primordial germ cellsurvival in culture, Nature 352, 809-811, 1991) and Matsui et al.(Effect of steel factor and leukemia inhibitory factor on murineprimordial germ cells in culture, Nature 353, 750-752, 1991). Solublesteel fragment refers to a fragment cleaved from the extracellulardomain at a specific proteolytic cleavage site. Membrane associatedsteel factor refers to both normal steel factor before it has beencleaved or the steel factor which has been altered so that proteolyticcleavage cannot take place. Steel factor is well known in the art; seeEuropean Patent Publication No. 0 423 980 A1, corresponding to EuropeanApplication No. 90310889.1.

[0039] The vector can be used to transfect many types of cells. Forexample cell lines and primary cells have been targeted successfully bythe present method. Examples of cell lines include, but are not limitedto MO7e cells, cultured cells include, but are not limited to NK cells,and primary cells include, but are not limited to peripheral T cells andperipheralized stem cells. Peripheralized stem cells are stem cells thathave been stimulated to leave the bone marrow and move into the blood,where they may be collected. In primary cells, the results using thebiotinylated virus linked via avidin to biotinylated targeting moietyshow a ten fold improvement in transfection over the control vector.Also, the favorable results with the NK cells is worthy of note, sinceNK cells are known to be very difficult to transfect. Having shown thatthe present vector is effective in a wide variety of cell types, thepresent invention has provided a vector that can be used in manydifferent cell types. Although hematologic cells have been emphasized,the invention is by no means limited to just hematologic cells.

[0040] The present invention further provides a method for targeteddelivery of a nucleic acid to a selected cell in a subject comprisingadministering to the subject a composition comprising a biotinylatedrecombinant adenovirus, wherein the recombinant adenovirus comprises thenucleic acid, wherein the virus is linked via streptavidin to abiotinylated targeting moiety that specifically targets the selectedcell.

[0041] The subject can be any animal, preferably a mammal, such as ahuman, a veterinary animal, such as a cat, dog, horse, pig, goat, sheep,or cow, or a laboratory animal, such as a mouse, rat, rabbit, or guineapig.

[0042] Any nucleic acid can be selected for use in the vector, and thustransferred into the target cell. For example, the GENBANK and EMBLdatabases can be searched to find genes/nucleic acid sequences desiredto use in a vector. The nucleic acid transferred into a targeted cell bya vector of the present invention can be a nucleic acid thatfunctionally encodes a protein or it can encode an oligonucleotide(e.g., antisense RNA, expression regulatory sequences). A nucleic acidencoding a selected protein can readily be determined based upon theamino acid sequence of the selected protein, and, clearly, many nucleicacids will encode any selected protein. The nucleic acid can comprise asequence that promotes cell-type specific expression (Wirak et al., EMBO10:289 (1991)). For example, to functionally encode, i.e., allow thenucleic acid to be expressed, the nucleic acid can include, for example,exogenous or endogenous expression control sequences, such as an originof replication, a promoter, an enhancer, and necessary informationprocessing sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Construction of such polynucleotides is well-known in the art and isdescribed further in Maniatis et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989). Preferred expression control sequences can be promoters derivedfrom metallothionine genes, actin genes, immunoglobulin genes, CMV,SV40, adenovirus, bovine papilloma virus, etc.

[0043] The promoter utilized can be any desired promoter, selected byknown considerations, such as the desired level of expression of anucleic acid functionally linked to the promoter and the cell type inwhich the vector is to be used. Promoters can be an exogenous or anendogenous promoter. Promoters can include, for example, known strongpromoters such as SV40 or the inducible metallothionein promoter, or anAAV promoter, such as an AAV p5 promoter. Additional examples ofpromoters include promoters derived from actin genes, immunoglobulingenes, cytomegalovirus (CMV), adenovirus, bovine papilloma virus,adenoviral promoters, such as the adenoviral major late promoter, aninducible heat shock promoter, respiratory syncytial virus, Roussarcomas virus (RSV), etc. To facilitate targeted expression of thenucleic acid in a particular tissue, a tissue specific promoter can beused in the vector. Examples of such promoters are known in the art.

[0044] The protein expressed by a cell transfected with the presentinvention can be isolated for use. The protein can be readily obtainedby any of several means. The coding regions of the transfected genes canbe expressed or synthesized, an antibody specific for the resultingpolypeptide can be raised by standard methods (see, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988), and the protein can be isolated fromother cellular proteins by selective interaction with the antibody. Thisprotein can be purified to the extent desired by standard methods ofprotein purification (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

[0045] Nucleic acids can also include sequences encoding antisenseoligonucleotides that can be transferred into cells to, upon expression,reduce or eliminate the expression of a gene, particularly associatedwith a disease or condition, typically by binding to mRNA encoded by thetarget gene. Nucleic acids can range in size, and will generally be from10 to 6000 nucleotides in length. The preferred length will depend onthe intended purpose of the nucleic acid and the viral vector; if toencode a protein or polypeptide, it will have a length determined by thenumber of amino acids to be encoded, and if to encode a antisensemolecule, it will have a length chosen to allow or maximizehybridization to the target nucleic acid (typically, 10 to 40nucleotides). If adenovirus is used, the nucleic acid can be up to about35 kilobases (kb). If AAV-2 is used the nucleic acid is considerablysmall, in the range of about 4.3 kb. Oligonucleotides having theselected binding specificity can readily be determined, as known in theart. For general methods relating to antisense polynucleotides, seeAntisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988).

[0046] The target can include a tissue, an organ, a cell, protein, apeptide, or uncharacterized markers and targets. For example, one cantarget cells of a tumor, metastatic cells, cells of the vasculature(such as endothelial cells and smooth muscle cells), cells of the lungs,muscle (such as smooth muscle cells, cardiac muscle, etc.), cells of thekidneys, blood cells (such as T-cells), cells of the bone marrow, suchas stem cells, cells of bone, neurons and related neurological cellssuch as glial cells, brain cells, liver cells, or precursors of anyselected cell, etc. by selecting a ligand, targeting motif or antibodyspecific for the tissue or cells, such as a targeting motif or domainthat specifically binds a cell surface receptor expressed on the cellsof the organ or tissue. The present vector can be targeted to themucosa. For example the target receptor can be an integrin, such asαvβ3, αvβ5, αIIbβ3, a growth factor receptor, a hormone receptor, acytokine receptor, and the like. The target receptor can be a growthfactor-dependent receptor (e.g., epidermal growth factor, nerve growthfactor, etc.). The target receptor can also be a ligand-dependentreceptor (such as a steroid receptor, thyroid hormone receptor, retinoicacid receptor, retinoid X receptor, TCCD (dioxin) receptor, fatty acidactivatable receptors, and the like) or a stimulus-dependent receptor(such as peroxisome proliferator-activated receptor). Many of thesereceptors or factors can be found listed in Parker, M. G. (1993) SteroidHormone Action (Oxford University Press, New York, pp. 210), in Tsai, M.J. & O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486, and in theGenBank database, which will contain additional receptors as well as thecomplete nucleotide sequences of the genes and cDNAs. The targetingmoiety is selected based on the cellular target, and it is routine tomake such a selection.

[0047] Depending upon the target, the present invention can be used intreatment of various conditions and diseases, to increase expression ofa desired protein, to inhibit expression or function of a gene product,etc. Any condition or disease in which transfer and expression of a genewould be beneficial, such as by reducing or eliminating symptoms of thedisease or condition can be treated by this method. For example, one cantreat leukemia, treat viral infections (e.g., HIV, HCV, HSV, HPV,hepatitis, influenza, etc.), reduce in size or eliminate or preventmetastasis of solid tumors that express a tumor-specific cell surfacemarker, such as melanoma, breast cancer, prostate and colon cancer amongothers. The vector of the invention can also be used to administernucleic acids encoding vaccine antigens for presentation of antigen bythe target cells.

[0048] Vectors of the invention can be administered to a subject or ananimal (e.g., for veterinary use or in animal model testing) by any ofmany standard means for administering the a composition. The vectors canbe administered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, topically,transdermally, to the mucosa, or the like. Vector can also be deliveredstereotactically for neural administration. Compositions can includevarious amounts of the selected vector in combination with apharmaceutically acceptable carrier and, if desired, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,etc. Parental administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Dosages willtypically be in the range of, for example, for direct injection into acell, tissue or organ, about 2×10⁷-2×10 ⁹ infectious particles; forintraperitoneal administration, about 10⁷, 10⁸, 10⁹ or up to 10¹²infectious particles; for inhalation into the airway, about 2×10⁹ pfu inabout a 20 ml volume; for direct instillation into the intrapleuralspace of the lung, about 1×10⁹ pfu, which can escalate to about 1×10¹¹;for stereotactic administration into the CNS, about 10⁸-10¹¹ infectiousparticles; and can be a dosage that can achieve protein levels of thetransfected gene, such as about 1-10 ng of reporter gene protein/mg cellprotein, as shown herein for luciferase.

[0049] Furthermore, the present invention can be used to administer avector into a cell ex vivo, such as for then transferring the cell intoa subject. For example, one can transfect a cell, such as a blood cell,a liver cell, a kidney cell, a neuronal cell, etc., that has beenremoved from a subject and, after transfection, transplant the cell backinto the patient. For example, one could harvest a subject's T cells,transfect them with a vector having an HIV antisense sequence, andreturn the cells to the patient to treat an HIV infection or to renderthe subject resistant to HIV invention. Alternatively, one can transfectcells derived from a donor subject or cultured cells and transplant thetransfected cell into the subject.

[0050] In general, for such an ex vivo administration, cells areisolated from a subject by standard means according to the cell type andplaced in appropriate culture medium, also according to cell type (see,e.g., ATCC catalog). The cells are then contacted with a vector asappropriate for the cell type, and the vector is allowed to transferinto the cells. Cells can then be transplanted back into the subject'sbody, by means standard for the cell type and tissue (e.g., in general,U.S. Pat. No. 5,399,346).

[0051] The present vectors can also be utilized to transfer a nucleicacid into a selected cell type in vitro. Such transfers can be utilizedfor a variety of purposes, such as to create a cell that can producelarge quantities of a selected protein, which can then be harvested. Thepresent vector and method can be particularly useful when the cellcomposition contains more than one cell type, and a specific cellpresent in the composition is to be targeted.

EXAMPLES

[0052] Production of Targeted Recombinant Viral Vectors

[0053] One example of the viral component of the vector is a recombinanthuman serotype 5 adenovirus (Ad5) which is replication deficient due toa deletion of the early 1 (E1) gene. Specifically, the gene for theenzyme firefly luciferase, under transcriptional control of the CMVpromoter and with an SV40 polyA addition sequence, was introduced intothe viral E1 coding region by homologous recombination (AdCMV-Luc).Replication defective AdCMV-Luc was propagated in the cell line 293,which carries the Ad5 E1 gene in its genome to compliment the deletionin AdCMV-Luc. Concentrated Adluc suspensions were produced by infecting293 cells with the virus, allowing time for the virus to replicate, andpurifying the virus from lysates of these cells. Specifically, adherent293 cells were grown to approximately 80% confluency in T150 flasks inMinimum Essential Media (MEM) containing 10% FBS and Pen/Strep (completeMEM). The media was then replaced with 10 ml MEM containing 2% FBS andthe cells were infected with Adluc at a multiplicity of infection (MOI)of between 0.1 and 1. Infected cells were incubated for 2 hours at 37°C. prior to adding 20 ml of complete MEM. Flasks were then incubated at37° C. until the cells became rounded from cytopathic effects of theviral infection, generally about 48 hours. Cells were detached by gentleshaking and collected by centrifugation at 200×g at 4° C. for 10minutes. The cells were lysed by 4 freeze/thaw cycles, the cellulardebris pelleted by centrifugation at 3500×g at 4° C. for 20 minutes, andthe supernatant containing the adenoviral particles was collected. Thissupernatant was concentrated and purified twice by ultracentrifugationover a discontinuous cesium chloride gradient of 1.33 and 1.45 mg/ml andthe apparent lower band collected. Cesium chloride was removed bypassing the suspension through a desalting column and the purified Adlucsuspension was then used in the vector production or stored at −70° C.after a 1:1 dilution in viral preservation media (10 mM Tris pH 8.0, 50mM NaCl, 0.1% BSA, 50% glycerol) was made.

[0054] Biotin was covalently linked to intact adenoviral particles usinga protocol for photoactivatable biotinylation. Briefly, 7.0 ml ofpurified Adluc suspended in Hepes buffered saline (HBS), with a titer of1.4×10¹¹ particles/ml, was treated in the dark with 100 μg/ml Immunopurephotoactivatable biotin (˜8×10⁵ biotins per virion), although as littlea 25 μg/ml or a much as 1000 μg/ml can be used. Five hundred microliteraliquots of the virus/photoactivatable biotin mixture were placed on icein open 1.0 ml Nunc tubes and the tubes irradiated at a wavelength of350 nm for 5 minutes in a laminar flow hood. Exposure of thephotoactivatable biotin to 350 nm light produces a reactive nitrinewhich then covalently links the biotin to any adjacent molecules.Although the reactive nitrine prefers to react with amino groups it is anon-specific binding process even capable of replacing C—H and C—Cbonds. The suspension was then passed through a PD-10 column containingSephadex G-25 (Pharmacia) to remove any unbound biotin and the effluentcontained the biotinylated recombinant adenovirus (bio-Adluc). Thisprocess can use other types of biotin, however it should be noted thatit is possible to overbiotinylate the virus to an extent that results inthe formation of insoluble complexes when avidin is added. Thisphenomenon was not encountered at the above concentrations of biotin.

[0055] Attachment of a biotinylated ligand to biotinylated recombinantadenovirus requires the addition of an avidin bridge to link the virusto the ligand. To add the avidin bridge, 500 μl of bio-Adluc in viralpreservation media, with a titer of 1×10¹⁰ particles/ml, was incubatedfor 30 minutes at room temperature with 2.5 μg Neutravidin (Pierce) fora final concentration of 5 μg/ml (˜1×10⁴ Neutravidin molecules/virion).Unbound Neutravidin was removed by gel filtration through a PD-10 columncontaining a 5 milliliter bed volume of Sepharcryl 300 (S-300)equilibrated with HBS.

[0056] Recombinant AAV is substituted in the above method to produce anAAV-based vector of this invention. Biotynylated AAV-2 has thus beenproduced, as above, with the exception that a different column is usedto purify the biotinylated virus. Likewise bitinylated retrovirus hasalso been made using the method described above, without significantreduction in infectivity.

[0057] Infection of Cells with Targeted Vector

[0058] The protocol for infecting MO7e cells with a SCF-targeted vectorwas as follows 1) A suspension of biotinylated recombinant adenoviruscontaining approximately 1×10¹¹ particles/ml in viral preservationbuffer was treated with 5.0 μg/ml Neutravidin for 45 minutes at roomtemperature to produce biotinylated recombinant adenovirus linked toavidin (control vector) 2) Five hundred μl volumes were filtered throughPD-10 columns containing a 5 ml bed of S-300 gel equilibrated with HBSto remove excess avidin 3) To make the SCF-targeted vector, biotinylatedrecombinant adenovirus linked to avidin was incubated with recombinanthuman bio-SCF at a concentration of 250 ng/ml for 30 minutes at roomtemperature 4) 1.5×10⁶ MO7e cells in 1.0 ml RPMI 1640 containing 2% seraand 30 ng/ml GM-CSF (infection media) were treated with 100 μl of theSCF-targeted or a control vector to obtain an MOI of between 75 and 2505) The virus/cell mixture was incubated in 6 well plates for two hoursat 37° C., then diluted with 2.0 ml RPMI media to obtain a finalconcentration of 10% FBS, 100 ng/ml SCF, 30 ng/ml GM-CSF and IL-3 andincubated for 24 to 48 hours (or longer as indicated) at 37° C. To assayfor luciferase activity, cells were collected, washed once with PBS andlysed with between 75 and 300 μl of lysis buffer containing 1% Triton-X100, 50 mm NaCl, 10 mM Tris pH 7.6 and 5 mm EDTA.

[0059] NK cells, peripheral T cells and peripheralized stem cells aretransfected by the present vector using the basic protocol describedabove.

[0060] Peripheralized Stem Cells: Peripheral blood (GM-CSF mobilized)were cryopreserved. We thawed them, separated the light density fractionon Ficoll gradients, and used these cells as targets for SCF directed,anti-CD34 and anti-CD44 directed infection. Using essentially the methodas described for MO7e cells, targeting was successful with anti-CD44vectors. Next, the cell population is enriched to target c-kit and CD44.

[0061] Primary T Cells: The cells were separated on Percoll gradient(yields >90% pure population of T-cells) and cultured 48 hours inabsence (unstimulated) and presence (stimulated) of 1 μg/ml PHA. Usingessentially the method as described for MO7e cell,. the cells were usedas targets for virus infection. Increases in infection of unstimulatedcells was observed with anti-CD44 (anti-CD3 moderately). Cells werestimulated with bio IL-2, anti-CD4, anti-CD8, anti-CD44, anti-CD5,anti-CD45, and anti-LFA-1α.

[0062] NK Cells: NK cell lines were used in an identical manner to thatdescribed for MO7e cells. MB02, KG1a, K562, HL-60 cell lines, etc. canalso be transfected using the same basic protoco as described. Positiveresults were obtained for targeting with anti-CD45, anti-LFA-1α,anti-CD44, bio IL-2.

[0063] Antibody-Targeted Vectors

[0064] To switch the specificity of vector targeting biotinylated,antibodies were substituted for the bio-SCF used in step 3 above. Allother portions of the protocol were unchanged. Specifically, to targetCD34 and CD117, biotinylated monoclonal antibodies to these antigenswere used at 20 μg/ml and to target CD44 the antibody was used at 2.5μg/ml.

[0065] Expression Assays

[0066] Luciferase assays were performed according to the protocolprovided with reagents purchased from Promega. Briefly, 20 μl of celllysate are mixed with 100 μl of luciferase assay reagent and the lightemission, in relative light units, determined on a luminometer using a10 second measuring time. Four consecutive 10 second measurements weremade and the average emission calculated.

[0067] Protein assays were performed on lysates using a Pierce BCAprotein assay. Briefly, 10 μl of the cell lysate is mixed with 200 μl ofthe BCA protein assay reagent, incubated for 30 minutes at 37° C. andthe protein content determined by comparison to BSA standards followingdetermination of the absorption of light at a wavelength of 595 nm on aspectrophotometer. Duplicate samples were evaluated and the averageprotein concentration calculated.

[0068] The luciferase activity in the cell lysate was determined bydividing the average emission determined on the luminometer by theaverage protein content in the sample to obtain a value reported asrelative light units per milligram of protein (RLU/mg).

[0069] Targeting and Expression Results

[0070] Results are presented in FIGS. 1-10. As can be seen, transfectionis specific and dependent upon the linkage of targeting moiety to theadenoviral vector.

[0071] In gene transfer to cell lines, experiments have been performedto assay the percentage of cells transfected by the present vectors thathave luciferase gene as the transferred DNA. Transfected cells werefixed and assayed individually for presence of luciferase DNA. Greaterthan 95% of cells were positive for the presence of luciferase DNA.Additionally, untransfected cells were assayed for background;background was found to be essentially zero.

[0072] Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

[0073] Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

What is claimed is:
 1. A composition for targeted delivery of a nucleicacid to a cell comprising a biotinylated recombinant adenovirus, whereinthe recombinant adenovirus comprises the nucleic acid, and wherein therecombinant adenovirus is linked via streptavidin to a biotinylatedtargeting moiety.
 2. The composition of claim 1, wherein the targetingmoiety is a ligand.
 3. The composition of claim 1, wherein the targetingmoiety is an antibody.
 4. The composition of claim 1, wherein thetargeting moiety is steel factor.
 5. The composition of claim 1, whereinthe targeting moiety is an antibody directed against cell surfacemarkers for steel factor.
 6. The composition of claim 1, wherein thetargeting moiety is antibody anti-CD34.
 7. The composition of claim 1,wherein the targeting moiety is antibody anti-CD44.
 8. The compositionof claim 1, wherein the targeting moiety is antibody anti-CD117.
 9. Thecomposition of claim 1, wherein the nucleic acid encodes a cytotoxicprotein.
 10. A method for targeted delivery of a nucleic acid to aselected cell in a subject comprising administering to the subject acomposition comprising a biotinylated recombinant adenovirus, whereinthe recombinant adenovirus comprises the nucleic acid, and wherein therecombinant virus is linked via streptavidin to a biotinylated targetingmoiety that specifically targets the selected cell.
 11. The method ofclaim 10, wherein the targeting moiety is a ligand.
 12. The method ofclaim 10, wherein the targeting moiety is an antibody.
 13. The method ofclaim 10, wherein the targeting moiety is steel factor.
 14. The methodof claim 10, wherein the targeting moiety is an antibody directedagainst cell surface markers for steel factor.
 15. The composition ofclaim 10, wherein the targeting moiety is antibody anti-CD34.
 16. Thecomposition of claim 10, wherein the targeting moiety is antibodyanti-CD44.
 17. The composition of claim 10, wherein the targeting moietyis antibody anti-CD117.
 18. The composition of claim 10, wherein thenucleic acid encodes a cytotoxic protein.
 19. A composition for targeteddelivery of a nucleic acid to a cell comprising a biotinylatedrecombinant encapsidated virus, wherein the recombinant virus comprisesthe nucleic acid, and wherein the recombinant virus is linked viastreptavidin to a biotinylated targeting moiety.
 20. The composition ofclaim 19, wherein the virus is an adenovirus.
 21. The composition ofclaim 19, wherein the virus is an adeno-associated virus.
 22. The methodof claim 19, wherein the targeting moiety is a ligand.
 23. The method ofclaim 19, wherein the targeting moiety is an antibody.
 24. The method ofclaim 19, wherein the targeting moiety is steel factor.
 25. The methodof claim 19, wherein the targeting moiety is an antibody directedagainst cell surface markers for steel factor.
 26. The composition ofclaim 19, wherein the targeting moiety is antibody anti-CD34.
 27. Thecomposition of claim 19, wherein the targeting moiety is antibodyanti-CD44.
 28. The composition of claim 19, wherein the targeting moietyis antibody anti-CD117.
 29. The composition of claim 19, wherein thenucleic acid encodes a cytotoxic protein.
 30. A method for targeteddelivery of a nucleic acid to a selected cell in a subject comprisingadministering to the subject a composition comprising a biotinylatedrecombinant encapsidated virus, wherein the recombinant virus comprisesthe nucleic acid, and wherein the recombinant virus is linked viastreptavidin to a biotinylated targeting moiety that specificallytargets the selected cell.
 31. The method of claim 30, wherein the virusis an adenovirus.
 32. The method of claim 30, wherein the virus is anadeno-associated virus.
 33. The method of claim 30, wherein thetargeting moiety is a ligand.
 34. The method of claim 30, wherein thetargeting moiety is an antibody.
 35. The method of claim 30, wherein thetargeting moiety is steel factor.
 36. The method of claim 30, whereinthe targeting moiety is an antibody directed against cell surfacemarkers for steel factor.
 37. The composition of claim 30, wherein thetargeting moiety is antibody anti-CD34.
 38. The composition of claim 30,wherein the targeting moiety is antibody anti-CD44.
 39. The compositionof claim 30, wherein the targeting moiety is antibody anti-CD117. 40.The composition of claim 30, wherein the nucleic acid encodes acytotoxic protein.